Cultivation of dha-rich prey organisms for aquatic species

ABSTRACT

A method is provided for producing prey organisms such as Artermia and rotifers, for feeding aquacultural organisms in particular at the larval stage. The method comprises cultivating the prey organisms during at least part of their life cycle in an aqueous medium comprising at least one lipid component having a DHA content of at least 30 wt %. The enriched prey organisms preferably have a DHA content of at least 12 wt % of their total lipid content. The prey organisms are suitable feed for larvae of fish including halibut, turbot, bass, and flounder, and crustaceans and molluscs.

FIELD OF THE INVENTION

The present invention is within the field of aquaculture, in particularthere is provided a method for producing prey organisms enriched inhighly unsaturated fatty acids (HUFAs), particularly docosahexaenoicacid (DHA). Also provided are feed compositions based on such organisms.

TECHNICAL BACKGROUND AND PRIOR ART

The consumption of seafood species for which there is a high consumerdemand such as salmon, trout, halibut and eel is increasing and due tothis high demand and limited natural stocks, much effort is spent ondeveloping cost effective aquacultural methods of farming such species.A particularly serious problem is to secure a high survival rate of thehatched larvae of the species being cultivated.

Expansion of the aquaculture industry requires that several problems beaddressed, one of the most significant being the difficulty of supplyinglive prey organisms which provide a nutritionally adequate feed for thelarvae. Larval fish in the wild consume a mixed population ofphytoplankton prey organisms that provide a balanced nutrition. However,collecting phytoplankton in sufficient quantities to meet the demand inaquaculture is not feasible. As an alternative, selected species of preyorganisms, in particular rotifers and Artemia species, are presentlycultivated and used as feed.

Generally however, such artificially cultivated prey organisms, althoughthey provide adequate amounts of protein and energy, have a lipidcomposition which is not adequate to cover the requirement for certainHUFAs, in particular DHA and EPA which are essential for the optimumsurvival, growth and development of larvae. Specifically, it has beenshown that a high content of DHA is required and that the ratio betweenDHA and EPA in the prey organisms should be at least 1:1 and preferablyat least 2:1. To provide prey organisms having such a composition inrespect of HUFAs it is necessary to cultivate the organisms in thepresence of enrichment compositions having a high content of DHA,preferably at least 20 wt % and a ratio of DHA to EPA exceeding theratio aimed at in the prey organisms, such as at least 3:1 andpreferably higher.

Currently, this problem is being addressed by cultivating the preyorganisms in the presence of enrichment compositions permitting theorganisms to be enriched in respect of these essential fatty acids.However, presently available commercial compositions for that purposesuch as emulsion products sold under the tradename Selco (TM) do notmeet the above requirements in that the DHA content is relatively lowand/or the DHA:EPA ratio is not high enough. Using such compositionsArtemia enrichment levels of 3-5% DHA of total lipids have been reported(McEvoy et al. Aquaculture 163 (1998) 237-250), and 12 to 15% survivalrates of fish fed such Artemia (McEvoy et al. supra; Navarro et al. J.Fish Biol. 43 (1993) 503-515). In this context, survival rates aredefined as survival percentage from the first feeding throughmetamorphosis. For cost-effective aquaculture production a larvalsurvival rate of 50% and preferably higher should be obtained.

Other commercially available compositions for prey organism enrichmentare products sold under the tradename Algamac (TM) containing up to 14wt % of DHA, and tuna orbital oil (TOO) that contains up to 30 wt % ofDHA.

WO 99/37166 discloses a method for the enrichment of live prey organismswith nutrients essential for fish larvae based on the use of dry soappowders of HUFAs obtained from the waste stream of marine algae oilextraction. The raw material for providing these powders has a contentof phospholipids and it contains about 23 wt % of DHA, but apparentlyvery little of other n-3 fatty acids. Artemia DHA enrichment levels ofabout 2.7% of dry weight are disclosed, but the use in aquaculture andefficacy with respect to predator larvae survival is not disclosed.

Another material intended for use in aquaculture is described in WO99/06585. Examples disclose a DHA content of 24 wt %, but thephospholipid content is not disclosed. The material however, contains ahigh proportion of free fatty acids (about 32-37 wt %) and a highcontent of non-lipid material (about 39-44 wt %), which may reduce thelipid uptake efficiency of prey animals. A high content of free fattyacids is generally considered harmful for fish larvae and juveniles.

Neither of the two last-mentioned materials is fish-based and they lackmany HUFAs found in fish, such as EPA and other n-3 fatty acids.

In a recent review by Sargent et al. (Aquaculture 179 (1999) 217-229) itis emphasized that in addition to the requirement in respect of HUFAs,fish larvae have a dietary requirement for phospholipids and it isstressed that the ideal diet for fish larvae is a diet having acomposition similar to the yolk of the eggs. According to these authorsfish egg yolk contains about 10 wt % (on a dry matter basis)phospholipids which contain about 17 wt % of DHA and about 9 wt % ofEPA. These authors conclude in their review that a problem remains withrespect to how to construct such a diet on a commercial scale fromcurrently available materials.

It has now been found that it is possible to provide—on a commercialscale—enriched aquacultural prey organisms having, in respect of HUFAsand phospholipids, a composition which is very close to that of fish eggyolk. By using the prey organisms of the invention it is possible tosecure optimum survival, growth, pigmentation and morphogenesis of fishlarvae such as halibut larvae. As demonstrated herein, the inventionprovides much higher survival rates during the larval stage andincreased quality parameters than previously disclosed for fish such asHalibut, thus making aquacultural rearing of many high-demand fishspecies more economical and commercially viable.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method of producing preyorganisms for use in aquaculture, in particular for feeding larval fish,the method comprising cultivating said organisms during at least part oftheir life cycle in an aqueous medium comprising at least one lipidcomponent having a DHA content of at least 30 wt %.

In a further aspect, the invention provides a composition for feedingfish in the larval and/or non-larval stage, the composition comprisingthe above mentioned prey organisms; said organisms having a content ofDHA of at least 12 wt % of the total lipid content of the organisms.

In yet a further aspect, the invention provides a method of producing acomposition having a water content that is less than 50 wt % includinghaving a water content less than 10 wt % comprising producing liveorganisms according to the aforementioned method and at least partiallyseparating the cultivated organisms from the aqueous medium.

In still a further aspect, the invention provides the use of the abovecompositions for feeding aquatic organisms at the larval and/orpost-larval stage.

DETAILED DESCRIPTION OF INVENTION

The method according to the invention, of producing prey organisms foruse in aquaculture, in particular for feeding larval fish, comprisescultivating said organisms during at least part of their life cycle inan aqueous medium comprising at least one lipid component having a DHAcontent of at least 30 wt %.

In the present context, the expression ‘prey organisms’ refers to anymarine organism which can be used as live feed for larvae of marinespecies which are produced in aquacultural facilities. A general reviewof such prey organisms can be found in Lavens & Sorgeloos (eds.) “Manualon the production and use of live food for aquaculture” published by FAO(1995) which is hereby incorporated by reference. Accordingly, the mostcommonly used prey organisms include several classes and genera ofmicroalgae, rotifers, Artemia, zooplankton including copepods,cladocerans, nematodes, and trochophora larvae.

As used herein, the term ‘aquaculture’ is to be understood in itsbroadest sense and it includes any production of any aquatic speciesproduced under aquacultural conditions, such as fish species, includingas examples salmon, trout, carp, bass, bream, turbot, sea bass, sole,milkfish, gray mullet, grouper, sea bream, halibut, flounder, Japaneseflounder, monkfish; crustaceans such as shrimp, lobster, crayfish andcrabs; molluscs such as bivalves.

A common feature of these aquatic species is that the life cycleincludes one or more larval stages which may have very specificnutritional requirements and accordingly the provision of live preyorganisms meeting this requirement is an essential factor for successfulaquacultural production. As mentioned above, one such specificrequirement is a high content of the essential fatty acid DHA, the term‘essential’ implying that the prey organisms are not capable of de novosynthesis of such compounds.

In a useful embodiment of the method, cultivation of the prey organismsis carried out until in at least 50% of the cultivated organisms the DHAcontent of their total lipid content is 12 wt % or higher. Preferablythe DHA content in the total lipid content of cultivated organisms is atleast 15 wt %, including at least 17.5 wt %, such as at least 20 wt %,including at least 25 wt %.

The current invention allows such prey animal cultivation on a largeeconomical scale, to provide suitable feed for successful commercialcultivation of fish species such as e.g. halibut, that are particularlysensitive during their larval stage and have proven very difficult toraise successfully in aquaculture with state of the art methods.

According to the method, the prey organisms to be cultivated may beselected from any prey organisms that can be raised and used inaquaculture, in useful embodiments the organisms are of a crustaceanspecies such as Artemia, Copepoda, Daphnia, or Moina species; a Rotiferaspecies including Brachionus plicatilis, Brachionus rotundiformis, andBrachionus rubens; or a Brachiopoda species.

The embodiments involving Artemia species are particularly useful. TheArtemia species can be cultivated and used according to the method ofthe invention at a naupliar, metanaupliar, or adult stage.

In specific embodiments of the method, the lipid component used is acomponent essentially comprising triglycerides or a phospholipid-richcomponent. As stated above, phospholipids are, in addition to DHAcontent, a critical ingredient for successful aquaculture rearing ofmany species. Therefore, in a useful embodiment of the method, theaqueous medium in which the organisms are cultivated comprises alipid-containing composition comprising a phospholipid-rich componentcomprising at least 25 wt %, such as e.g. at least 40 wt % ofphospholipids, preferably at least 50 wt % of phospholipids such as atleast 60 wt %, or at least 70 wt % phospholipids; the composition havinga DHA content of at least 30 wt %.

In the present context the term ‘phospholipids’ is used to describe aclass of lipids containing phosphoric acid as a mono- or diester. Thus,phospholipids include phosphatidyl choline (PC), phosphatidylethanolamine (PE), phosphatidyl inositol (PI), phosphatidyl glycerol(PG), phosphatidyl serine (PS), and phosphatidic acid (PA). The term‘lecithin’ is also commonly used for mixtures of the abovephospholipids.

In accordance with the invention any phospholipid-rich component can beused in the composition of the method. However, presently preferredphospholipids are phospholipids isolated from marine organism materials,including fresh materials and dried materials. Fresh materials includefor example viscera from fish and other marine animals, flesh of fish,fish eggs, squids, molluscs and a planktonic biomass. Dried materialsinclude, in particular, fishmeals such as meals of herring, capelin,mackerel, menhaden, sardine, anchovy, horse mackerel, blue whiting, andmeals of planktonic organisms.

Such fish-based materials are particularly preferred according to theinvention, as they provide not only a high content of DHA but they alsocontain EPA and other n-3 PUFAs, characteristic of fish, including 18:3,18:4, 20:4 and 22:5, and are to an extent similar to the natural diet offish larvae.

In useful embodiments, the phospholipid-rich component provides therequired amount of DHA. However, in other embodiments the DHA content ofthe phospholipid component may not be sufficient. In such cases, atleast one further lipid component that provides an adequate amount ofDHA must be incorporated. Typically, such a further lipid componentcontains at least 20 wt % of DHA, preferably at least 30 wt % of DHA,more preferably at least 40 wt % of DHA, for example at least 50 wt % ofDHA. In preferred embodiments, the further lipid component includes atleast 60 wt % of DHA, such as at least 70 wt % DHA, including at least90 wt % DHA.

If required, the further DHA-rich lipid component is incorporated in thecomposition of the invention in an amount that at least results in atotal DHA content of the composition which is at least 30 wt %.Depending on the DHA content of the phospholipid-rich component, theamount of the further lipid component required may be in the range of5-99 wt %. In certain embodiments of the invention the amount of thefurther lipid component is in the range of 50-95 wt %, such as in therange of 50-70 wt %, or higher.

In cases where it is required to supplement the phospholipid-richcomponent to enhance the level of DHA, the proportion of that componentmay vary in the range of about 1-99%, such as in the range of 2-75 wt %including the range of 5-50 wt %, such as 5-25 wt %, including the rangeof about 10-20 wt %.

The source of the further lipid component may be any naturally occurringlipids containing at least 20 wt % of DHA and any such lipidssynthesized chemically or enzymatically. In useful embodiments thefurther lipid component comprises essentially glycerides such astriglycerides. Such a component with a high content of DHA ca beprovided as a triglyceride by contacting DHA as a free fatty acid andglycerol in the presence of chemical catalysts or an enzyme capable offorming glycerides from the reactants, such as a lipase including alipase isolated from Candida antarctica. Free fatty acids though aregenerally not recommended as a major component in feed compositions forfish prey organisms, as it is generally considered in the state of theart, that a high free fatty acid content may be harmful for fishjuveniles. In preferred embodiments of the method of the invention, thelipid-containing compositions for feeding prey-animals contain less thanabout 10 wt % free fatty acids, and preferanly less than about 5 wt %,such as less than about 2 wt %. In certain embodiments, fish-basedsources are used for obtaining the further lipid component for thereasons mentioned above, i.e. that such materials additionally provideother desired n-3 HUFAs.

As mentioned above, it has been shown that not only a high content ofDHA is required in the feed for fish larvae, but the ratio between DHAand EPA in the prey organisms is of significance for the survival anddevelopment of the larvae of the cultured species. It is generallyrecognized that the DHA:EPA ratio in the prey organisms should be atleast 1:1 and preferably at least 2:1. In order to achieve this desiredratio in the prey organisms, a significantly higher ratio may berequired in the enrichment composition for the prey organism. Thus,according to the method of the invention the lipid-containingcomposition has in preferred embodiments a DHA:EPA weight ratio in therange of 1:1 to 10:1, more preferably 2:1 to 8:1, including the range of4:1 to 6:1.

As can be understood based on the above, a high total content of DHA inthe feeding composition of the method is of significance to achieve thedesired results of the invention, such as at least 30 wt %, preferablyat least 35 wt %, including at least 40 wt %, and more preferably atleast 50 wt % including at least 60 wt %, such as at least 70 wt %.

The concentration of the lipid component or the lipid composition in theaqueous medium of the method may be any useful concentration thatprovides effective feeding and thus enrichment of the prey organism. Inuseful embodiments this amounts to a concentration in the range of0.01-5 g/L of the lipid component or the lipid composition, includingthe range of 0.01-1 g, such as the range of 0.01-0.1 g/L.

In a preferred embodiment, the lipid component or lipid composition isin the form of an emulsion in the aqueous medium, such as in the form ofmicellar particles of size that allows for ingestion by the preyorganisms, e.g., such that the average largest dimension is in the rangeof 1-100 μm, including the range of 1-50 μm.

In certain useful embodiments the aqueous medium is sea water orsimulates sea water, with the addition of, e.g., sodium chloride.

In a further aspect, the present invention provides a composition forfeeding marine organisms at the larval and/or non-larval stage, thecomposition comprising prey organism having a content of DHA of at least12 wt % of the total lipid content of the organisms, preferably 15 wt %of the total lipid content, more preferably 20 wt % of the total lipidcontent, including at least 25 wt % such as at least 30 wt % and alsoincluding at least 35 wt % of the total lipid content. The organisms maybe any useful prey organisms that can be raised in aquaculture,including Crustacea species such as Artemia, Copepoda, Daphnia, andMoina; Rotifera species including Brachionus plichatilis; Brachiopodaspecies and Nematoda species.

As can be inferred from the above, in order for the composition toprovide a significant DHA ration the total lipid content in the preyorganisms needs to be significant, such as, e.g., at least 20 wt % on adry matter basis, and preferably at least 30 wt % of dry matter. Asmentioned, a high content of free fatty acids is not desirable in feedfor fish larvae and other aquacultivated organisms. Preferably, freefatty acids are less than about 10 wt % of total lipid of the preyorganisms according to the method of the invention.

In a particular embodiment, the composition comprises an aqueous phaseof at least 50 wt % which may comprise a salt such as sodium chloride,in a concentration such as at the least 0.5 wt %. The composition canalso be in a dry or semi-dry form such as less than 50 wt % of water,including less than 10 wt % of water, such as less than 1 wt % of water,such as essentially containing no water. The dry or semi-dry compositioncan be provided in the form of a powder, as granules, or as flakes.

Compositions comprising Artemia have been found to be particularlyuseful. The Artemia can be at a naupliar, metanauplii, or adult stage.

In yet a further aspect, the invention provides a method of producing acomposition as described above comprising at least partially separatingcultivated prey organisms as listed earlier from the aqueous medium inwhich they are cultivated. Particular embodiments of the method comprisethe step of drying the separated organisms to obtain the composition ina form as described above.

In a still further aspect, the invention provides the use of acomposition as described above for feeding aquatic organisms at thelarval and/or post-larval stage. The aquatic organisms can be anyspecies that can be cultivated aquaculturally, including salmon, trout,carp, bass, bream, turbot, sole, milkfish, gray mullet, grouper,flounder, sea bass, sea bream, cod, haddock, Japanese flounder, eel;crustaceans such as shrimp, lobster, crayfish and crabs; molluscs suchas bivalves.

In certain embodiments, the invention provides the use of a compositionfor feeding aquatic organisms raised not primarily for consumption, suchas ornamental fish species and aquarium fish species.

EXAMPLE 1

Large Scale Isolation of a Phospholipid-Rich Lipid Component from SquidMantles

Minced squid (150 kg) was added to 300 L of isopropanol and the mixturewas agitated rather vigorously for 4-6 h and left to stand overnight.Subsequently, the mixture was filtered and 300 L of hexane were added tothe filtrate and mixed. This resulted in two phases which were allowedto separate. The upper phase, which largely consisted of hexane andisopropanol was separated and subjected to distillation in severalrounds in vacuum using a 50 L rotary evaporator to yield a total of 2.2kg of a phospholipid enriched fraction as a brown-yellowish wax having aphospholipid content of about 65 wt % and the following total fatty acidcomposition (numbers in left-most column refer to the number of carbonsand double bond in the fatty acids of the lipid components, DHA is 22:6and EPA 20:5):

14:0 1.9 16:0 28.3  16:1 0.6 18:0 2.9 18:1 3.2 18:2 0.2 18:3 0.0 18:40.2 20:1 2.7 20:4 1.4 20:5 13.8  21:5 0.0 22:1 0.0 22:6 40.4  95.5 

EXAMPLE 2

Preparation of an Enrichment Composition for Fish Larvae Prey Organisms

A composition for prey organisms such as Artemia species was prepared bycombining and mixing the following ingredients:

TABLE 2.1 phospholipid-rich component from squid mantles 9.7 g(Example 1) TG 4010 (TM), Croda, essentially triglycerides 78.0 g w/≈ 40wt % DHA vitamin C (ascorbyl palmitate) 8.5 g co-emulsifer, BASFChremophore A25 (TM) 1.6 g Glucan Macroguard (TM) (immunostimulant) 0.8g vitamin A (vitamin A palmitate, 1 mill i.u./g) 0.190 g vitamin E(DL-alpha tocopherol acetate) 0.155 g vitamin B (thiamine hydrochloride)1.2 g TBHQ (antioxidant) 0.036 g Ethoxyquin (antioxidant) 0.036 g Total100 g

The TG 4010 material used as a DHA-rich component in the composition isderived from fish oil-based material which is enriched for DHA, itcomprises 40 wt % DHA, about 10 wt % EPA and about 10 wt % other n-3HUFAs. The fatty acids are mostly in the form of triglycerides and thematerial has a very low free fatty acid content. Other materials havebeen tested as sources of a DHA-rich component, such as TG 5010 (alsofrom Croda) which has a DHA content of about 50 wt %, and enzymaticallyhighly DHA-enriched triglycerides.

EXAMPLE 3

Use of Enrichment Composition for Cultivating Artemia

Artemia cysts were hatched under optimal conditions (in seawater, 27-29°C., pH about 8, oxygen content above 4 mg/L). The newly hatched naupliarArtemia were rinsed and transferred in 250 L tanks to give a density of200.000/L. Temperature was kept at 25-28° C., oxygen content at 5-6 mg/Land pH buffered at 7.5 with sodium bicarbonate (2 g/L). The tanks wereaerated by passing atmospheric air through perforated hoses at bottom oftanks. Enrichment composition as described in Example 4 was added to thetanks to a concentration of 0.2 g/L and the same amount added 10 hlater. 24 h after the first addition of enrichment composition theArtemia has the following lipid composition (31% dw (dry weight)lipids):

PL TG FFA Total 16% 76% 8% 100% 14:0 8.8 1.0 3.1 0.8 16:0 15.0  8.836.0  11.1  16:1 2.6 3.2 3.1 2.5 18:0 6.4 2.7 6.3 4.2 18:1 25.2  15.6 13.0  17.1  18:2 4.2 3.5 1.8 3.3 18:3 13.2  19.2  6.5 14.7  18:4 2.2 3.11.7 2.4 20:1 1.6 1.0 0.0 0.9 20:4 2.8 2.1 0.0 2.2 20:5 12.5  10.2  4.49.5 22:1 0.0 0.0 0.0 22:4 0.0 1.1 0.0 1.2 22:5 0.0 1.0 0.0 1.1 22:6 4.620.0  14.8  18.9  99.0  92.5  90.7  90.0 

The Artemia thus obtained has a highly enriched total concentration ofDHA in accordance with the invention and is thus particularly suitablefor feeding fish larvae such as halibut larvae.

EXAMPLE 4

Use of Enrichment Composition for Cultivating Artemia

Newly hatched Artemia were placed in 250 L tanks and same conditions asdescribed in Example 3. The Artemia were fed a lipid composition mixed 2wt % Chremophore A25 emulsifier. The lipid composition contained 50 wt %phospholipid composition of Example 1; 25 wt % ‘DHA-80’, essentiallytriglycerides comprising 80 wt % DHA, synthesized enzymatically fromglycerol and DHA fatty acid using lipase from Candida Antarctica (asdescribed in U.S. Pat. No. 5,604,119); and 25 wt % Lysi-22 (TM) (Lysihf, Iceland), a fish oil with 22 wt % DHA. The feed composition wasadded to the tanks to a concentration of 0.2 g/L and the same amountadded 12 h later. 24 h after the first addition of enrichmentcomposition the Artemia has the following lipid composition (34% dwlipids):

PL TG FFA Total 25% 72% 3% 100% 14:0 0.9 1.1 0.0 1.3 16:0 13.6 10.6 32.011.2 16:1 3.3 3.5 3.3 3.4 18:0 5.8 2.2 10.6 3.3 18:1 26.2 15.4 15.7 15.118:2 3.7 2.7 0.0 2.5 18:3 13.8 15.0 4.4 13.7 18:4 2.7 2.1 0.0 2.2 20:11.0 1.9 5.3 2.0 20:4 2.1 1.7 0.0 1.9 20:5 13.1 8.7 5.0 9.7 22:6 8.4 28.823.6 28.0 94.6 93.7 100.0 94.1

The Artemia obtained has a very highly enriched total concentration ofDHA (9.5 wt %) in accordance with the invention as well as otherfish-characteristic n-3 HUFAs, and is thus particularly suitable forfeeding fish larvae such as halibut larvae.

EXAMPLE 5

Use of Enrichment Composition for Cultivating Rotifers (Brachionusplichatilis)

Rotifers were reared under similar conditions as described in Example 3,they were fed with Isochrysis plankton and yeast and enriched for 6 h at27 C with an enrichment composition as described in Example 2, exceptthat Croda 50 was used instead of Croda 40, Croda 50 containing about 50wt % of DHA. The rotifers had the following lipid composition (22% dwlipids):

PL TG FFA Total 32% 56% 13% 100% 14:0 6.6 7.8 3.3 6.9 16:0 25.9  4.915.2  13.0  16:1 1.9 2.5 1.3 2.2 18:0 3.6 5.7 2.7 4.7 18:1 4.5 4.5 5.54.7 18:2 4.9 0.3 2.0 2.0 18:3 3.1 3.2 1.9 3.0 18:4 2.2 6.2 2.2 4.4 20:11.2 1.9 1.5 1.6 20:4 5.0 2.3 2.2 3.2 20:5 10.1  14.7  14.8  13.4  22:625.6  38.8  40.4  35.2  94.7  92.7  93.0  94.3 

The rotifers obtained have a very high total concentration of DHA,distributed in the different lipid classes analyzed and thus exemplifiesthe efficacy of the invention.

EXAMPLE 6

Comparison of Enrichment Compositions for Cultivating Artemia

Artemia cysts hatched as in Example 3 and transfer to cultivation tankswhere conditions were kept as in Example 3 (except for some differencein temperature, see table). Enrichment compositions were preparedsimilar as described in Example 2, i.e. with same additives added as inTable 2.1 such as emulsifier, vitamins and also about 10% of thephospholipid-rich component from squid mantles as described inExample 1. The bulk ingredient (about 80%) of the preparations werecommercial lipid compositions as listed in Table x. These are AlgaMac2000 (TM), DHA Selco (TM), DC DHA (TM) and feed grade Cod Liver Oil(from Lysi, Iceland) The preparations were added to the tanks to aconcentration of 0.2 g/L and the same amount added 10 h later. 24 hafter the first addition of enrichment composition the Artemia has thefollowing lipid composition:

composition AlgaMac 2000 DHA Selco DC DHA Cod liver oil from Ex. 2 Tduring growth 20° C. 27° C. 27° C. 20° C. 27° C. % dw lipids 17% 24% 22%23% 31% 14:0 2.3 3.4 1.1 3.2 0.8 16:0 12.6 13.5 10.9 16.0 11.1 16:1 4.44.6 3.7 6.2 2.5 18:0 4.7 5.5 4.6 4.5 4.2 18:1 19.2 24.8 34.5 25.8 17.118:2 3.6 5.6 6.9 4.4 3.3 18:3 23.1 28.4 17.9 21.1 14.7 18:4 3.9 4.8 2.84.3 2.4 20:1 0.4 0.9 2.8 0.9 20:4 1.3 1.3 2.2 20:5 4.4 5.3 6.3 6.0 9.522:1 22:4 2.8 0.6 1.2 22:5 0.6 1.1 22:6 7.8 4.1 8.3 2.9 18.9 90.3 100.089.3 97.3 90.0

The Artemia enriched with the preferred composition according to theinvention has clearly a higher enriched total concentration of DHA inaccordance with the invention and is thus particularly suitable forfeeding fish larvae such as halibut larvae.

EXAMPLE 7

Use of HUFA- and Phospholipid-Enriched Artemia for Aquacultural Rearingof Halibut

Halibut larvae were first fed at 230-250° d. (‘° d’: multiplicationfactor of temperature (° C.) and days since hatching.) Circular rearingtanks were used, either 3.5 or 7 m³. Larvae were gradually acclimatizedto a rearing temperature of 11° C. and a light intensity of 300-500 lux.The larvae were fed Artemia twice per day, in the morning and in thelate afternoon. The Artemia was enriched with an enrichment compositionaccording to the invention 24 h before the morning feed, then stored at13-15° C. for another 7-8 h for the afternoon feed. Feed rations wereadjusted to allow for good digestion of the Artemia. Microalgae(Isocrysis sp.) were added to the rearing water to reduce stress andfacilitate maximum ingestion rates. Slight aeration was applied in thecenter of the tanks to homogenize the water quality and the feedparticles. Slight circular current was acquired with the inflow todistribute the larvae. Water exchange was increased from 1.2 times per24 h in the beginning up to 3.3 times per 24 h in the end. Larvalrearing tanks were cleaned daily.

Survival rates of over 80% in one tank from start of feed to end oflarval stage were observed (90% excluding “gapers”: larvae with jawdeformity), and frequently survival rates between 65 and 75% have beenobserved. On average about 80% of juveniles showed correct pigmentation,but up to 96% correct pigmentation in one tank were observed. Correctpigmentation is defined as a normal pigmentation color on the ocularside and no pigmentation on the blind side. About 65% of juveniles onaverage but up to 80% in one tank showed correct eye migration, that ishaving both eyes on the ocular side. Ongoing experiments indicate thateven higher average survival and pigmentation rates are obtainable.

The results show that DHA-enriched prey organisms according to theinvention are particularly suitable for the rearing of aquatic speciessuch as halibut in terms of high survival rates and quality.

What is claimed is:
 1. A method of producing prey organisms for use inaquaculture, the method comprising cultivating said organisms during atleast part of their life cycle in an aqueous medium comprising a feedcomposition having a docosahexaenoic acid (DHA) content of at least 30wt % and a ratio of DHA to eicosapentaenoic acid (EPA) which is at least3:1, the composition comprising a 2-75 wt % of a marine animalphospholipid-rich component comprising at least 25 wt % phospholipids;about 5-about 99 wt % of a further lipid component obtained fromfish-based sources; and less than 10 wt % of free fatty acids.
 2. Amethod according to claim 1 wherein the cultivation is carried out untilin at least 50% of the cultivated organisms the DHA content of theirtotal lipid content is 12 wt % or higher.
 3. A method according to claim2 wherein the DHA content of the total lipid content of the cultivatedorganisms is at least 15%.
 4. A method according to claim 2 wherein theDHA content of the total lipid content of the cultivated organisms is atleast 17.5 wt %.
 5. A method according to claim 2 wherein the DHAcontent of the total lipid content of the cultivated organisms is atleast 20 wt %.
 6. A method according to claim 1 wherein the organisms tobe cultivated are selected from the group consisting of a planktonicspecies, crustacean species, Rotifera species and Brachiopoda species.7. A method according to claim 6 wherein the crustacean species is anArtemia species.
 8. A method according to claim 7 wherein the Artemiaspecies is cultivated in the aqueous medium at a naupliar, metanaupliar,or adult stage.
 9. A method according to claim 6 wherein planktonicspecies includes a Rotifera species or a Brachiopoda species.
 10. Amethod according to claim 9 wherein the Rotifera species includesBrachionus Plicatilis, Brachionus rotundiformis, or Brachionus rubens.11. A method according to claim 6 wherein the crustacean species includeArtemia, Copepoda, Daphnia, or Moina species.
 12. A method according toclaim 1 wherein the phospholipid-rich component comprises at least 50 wt% of phospholipids.
 13. A method according to claim 1 wherein thephospholipid-rich component comprises at least 70 wt % of phospholipids.14. A method according to claim 1 wherein the further lipid componenthas a content of DHA of at least 30 wt %.
 15. A method according toclaim 14 wherein the further lipid component has a content of DHA of atleast 50 wt %.
 16. A method according to claim 1 wherein the amount ofthe further lipid component is about 50-about 95 wt %.
 17. A methodaccording to claim 1 wherein the amount of the phospholipid-richcomponent is about 5-about 50 wt %.
 18. A method according to claim 1wherein the total content of DHA in the composition is at least 40 wt %.19. A method according to claim 18 wherein the total content of DHA inthe composition is at least 50 wt %.
 20. A method according to claim 18wherein the total content of DHA in the composition is at least 60 wt %.21. A method according to claim 1 wherein the further lipid component isessentially triglycerides.
 22. A method according to claim 21 whereinthe essentially triglycerides component is obtained by contactingglycerol and DHA in the presence of a catalyst or an enzyme.
 23. Amethod according to claim 22, wherein the enzyme is a lipase.
 24. Amethod according to claim 22, wherein the enzyme is a lipase produced byCandida antarctica.
 25. A method according to claim 1 wherein thephospholipid-rich component is obtained by separating it from a marineorganism material.
 26. A method according to claim 25, wherein themarine organism material includes squid, fish eggs, planktonic biomass,or fish meal.
 27. A method according to claim 1 wherein the aqueousmedium comprises 0.01 to 5 g/L of a lipid composition.
 28. A methodaccording to claim 1 wherein a lipid composition is emulsified in theaqueous medium.
 29. A method according to claim 28 wherein said lipidcomposition is in the form of micellar particles having an averagelargest dimension of about 1-about 100 μm.
 30. A method according toclaim 28 wherein said lipid composition is in the form of micellarparticles having an average largest dimension of about 1-about 50 μm.31. A method according to claim 1 wherein the aqueous medium is seawater.
 32. A method according to claim 1 wherein the aquaculture isfeeding of larval fish.
 33. A method according to claim 1 wherein thefurther lipid component has a content of DHA of at least 40 wt %.
 34. Amethod according to claim 1 wherein the further lipid component consistsessentially of glycerides.
 35. A method according to claim 1 wherein atleast one of the DHA, EPA, or the further lipid component is in the formof a glyceride.
 36. A method according to claim 35 wherein the glycerideis produced by contacting at least one of DHA, EPA or the further lipidcomponent, as a free fatty acid, and glycerol in the presence of asuitable chemical catalyst or an enzyme.
 37. A method according to claim35 wherein at least one of the DHA, EPA or the further lipid componentis in the form of triglycerides.
 38. A method of producing a compositionfor feeding aquatic organisms in the larval stage, the non-larval stage,or a combination of the larval and non-larval stages, the compositioncomprising less than 50 wt % water and cultivated organisms selectedfrom the group consisting of a Crustacea species; Rotifera species;Brachiopoda species; and Nematoda species; said cultivated organismsincluding a content of docosahexaenoic acid of at least 12 wt % of thetotal lipid content of the organisms, and including a total lipidcontent of at least 20 wt % on a dry weight basis, the method comprisingproducing cultivated organisms according to the method of claim 1 and atleast partially separating the cultivated organisms from the aqueousmedium.
 39. A method according to claim 38 comprising the further stepof drying the separated cultivated organisms.
 40. A method according toclaim 38 wherein the Crustacea species include Artemia, Copepoda,Daphnia, or Moina species.